We have examined the complex interactions needed for generating an infectious virus. We showed that the mature capsid proteins from two different lentiviruses, HIV-1 and SIVmac, can assemble into the mature core of the infectious particle. Additionally, we quantified the amounts of functional nucleocapsid (NC) and p6 protein needed during HIV-1 infection, and determined the effects of treating HIV-1 with suboptimal concentrations of protease inhibitors on viral protein processing and RNA maturation. We are studying the trafficking of HIV-1 macromolecules and assembly. Once exported from the nucleus, HIV-1 RNA needs to traffic to proper subcellular locations to be translated or packaged; similarly, the newly synthesized Gag needs to be targeted to the plasma membrane for virus assembly. Using our newly developed imaging tools, we are investigating the mechanisms used by HIV-1 RNA for transport in the cytoplasm. HIV-1 RNA traffics to the plasma membrane for assembly; we will investigate the properties of this targeting event, including whether monomeric or dimeric RNA is targeted during assembly and the dwell time of the RNA that stays near the plasma membrane. We are currently testing the hypothesis that Gag-dimeric RNA interaction is the nucleation point of retroviral assembly. We are investigating Gag and RNA targeting in polarized T cells and in viral synapses. These experiments will provide an understanding of aspects of HIV-1 replication that we currently know very little about and will allow us to better understand how HIV-1 uses the cellular machinery to traffic its macromolecules. [Corresponds to Hu Project 3 in the October 2011 site visit report of the HIV Drug Resistance Program]